This proposal aims to continue the work of the original grant that investigated differentiation functions of cdk6. Recent evidence in the field suggests that cdk6 is an early target of pluripotency, suggesting it both promotes self-renewal and blocks differentiation, characteristics of pluripotent cells and cancer stem cells. Our lab has identified a novel interaction between cdk6 and the developmentally important transcription factor, Eya2. We have evidence that cdk6 squelches Eya2-driven transcriptional activation and we have developed an innovative approach to studying cdk6 using ovarian cancer tissue culture cells as a model system. The goal of the work outlined in this proposal is to investigate novel functions of cdk6 that contribute to the regulation of differentiation and oncogenesis. Important to this proposal is the fact that the cdk6 oncogene is emerging as one of the early targets of the major regulators of pluripotency and self-renewal. It has been shown to be a direct target of the three major regulators of pluripotency SOX2, NANOG and OCT4 (Bonini et al. 1998;Zhang et al. 2009). Thus, an understanding of the interaction between cdk6 and Eya2 could impact our understanding of the biology of both cancer stem cells and development. The relevant model system for this proposal is the ovary, where Eya2 has a demonstrated effect in Drosophila mutants (Bonini et al. 1998) and ovarian cancer cell lines, where Eya2 is elevated (Zhang et al. 2009). Elevated Eya2 has been associated with poor outcomes in ovarian cancer (Zhang et al. 2009). We envision a model in which cdk6 and Eya2 interact to affect the timing of cell cycle and differentiation that impacts development and transformation. Aims proposed here makeup a pilot research project that examines the biological relevance of this novel interaction. These Aims include 1) To confirm the interaction of cdk6 and Eya2 in an ovarian cancer cell line. To confirm that cdk6 influences the transcriptional activation function of Eya2 and determine what functions of cdk6 are required for this affect. To perform bandshift, ChIP and ChIP-seq studies to examine the DNA-binding and promoter occupancy of the Eya2/Six4 heterodimer. Aim 2) To isolate cdk6 mutants that disrupt binding of cdk6 to Eya2 and other proteins identified in a yeast two-hybrid screen. This genetic screen will utilize a pool of cdk6 mutants to search for mutants that disrupt or enhance binding to Eya2. Aim 3) To determine the functional consequences of the cdk6 Eya2 interaction using a variety of assays including immunofluorescence and kinase/phosphatase assays. To determine the effect of over-expression of Eya2, cdk6, and mutant versions of these proteins, on proliferation, migration, and tumorigenesis of ovarian cancer cells. The Aims presented here address the hypothesis that cdk6 and Eya2 interact to influence cell proliferation and differentiation-processes important in development and cancer-by examining the biological relevance of the novel interaction of cdk6 and Eya2, which was identified in our laboratory. PUBLIC HEALTH RELEVANCE: The goal of the work outlined in this proposal is to investigate the interaction of the cell cycle regulatory protein, cdk6 and the developmentally important transcription factor, Eya2. An understanding of how these proteins influence differentiation and cell proliferation is crucial to the biology of cancer stem cells and development and may advance our understanding ovarian cancer.